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AHN, Chang-Soon, 1941INTERSPECIFIC HYBRIDIZATION AMONG FOUR
SPECIES OF THE GENUS VIGNA SAVIo
University of Hawaii, Ph.D., 1976
Agriculture, plant culture
Xerox University Microfilms,
Ann Arbor, Michigan 48106
INTERSPECIFIC HYBRIDIZATION AMONG FOUR SPECIES
OF THE GENUS VIGNA SAVI
A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE
UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN HORTICULTURE
DECEMBER 1976
By
Chang-Soon Ahn
Dissertation Committee:
Richard W. Hartmann, Chairman
Gerald D. Carr
Har~yuki Kamemoto
Henry Y. Nakasone
Yoneo Sagawa
ABSTRACT
Crosses with three lines of mung bean
each of adzuki bean
(y.
(!.
(y.
angu1aris), black gram
radiata) and two lines
(!.
mungo) and rice bean
umbe11ata) were made in order to determine the barriers to hybrid-
ization between species, and to assess the possibility of overcoming
these barriers.
This would provide useful information for exploiting
wide germp1asm resources in breeding these valuable pulse crops.
Reciprocal differences were common in all the species combinations.
These differences were observed in pod set, embryo abortion, embryo
culture, and seedling lethality.
No crosses were successful in both
directions.
The results of six interspecific combinations are summarized as
follows:
(1)
tion.
Adzuki bean and black gram was the least successful combina-
Although pod set was normal when adzuki bean was used as the
female, embryo abortion was complete and no seedlings obtained from
embryo culture survived beyond the seedling stage.
(2)
Black gram and rice bean was also a very difficult combina-
tion to produce.
Some seedlings obtained by embryo culture from
crosses using black gram as the pistillate parent reached adult stage,
but died before flowering.
(3)
Adzuki bean and mung bean was a somewhat more successful
combination.
Hybrids from mung bean produced by embryo culture
flowered profusely but were completely sterile.
Lack of chromosome
pairing, shown by an average of 2.3911 + 17.221 seems likely to be a
principal cause of sterility.
iv
(4)
The mung bean and rice bean combination was somewhat easier
tomake than the last.
Embryo abortion is not complete so that some
crinck1ed but viable seeds are produced on mung bean used as the pistillate parent.
However, the hybrids, with average chromosome pairing
of O.09 1V + 4.69 11 + 12.26 1 , are still completely sterile.
Colchi-
cine-induced amphidiploids showed regular meiosis with 22 bivalents
and greatly improved fertility, indicating that at least in this
combination much of the sterility of the diploid hybrid is caused by
chromosomal factors.
However, low pod set and defective seeds in the
amphidiploid indicate that some adverse genic interactions are still
found.
(5)
The mung bean and black gram combination (the former used as
the pistillate parent) produced hybrid seeds with good germination.
However, the FI hybrids were only partially fertile.
One quadrivalent
in 37% of the cells examined indicates that one reciprocal translocation
has occurred during chromosome differentiation of these two species.
Segregation for morphological characters and the
appe~rance
of highly
fertile individuals in the F2 and BCl demonstrate the possibility of
gene exchange between these two species.
(6)
The adzuki bean and rice bean combination obtained by culture
of embryos from rice bean used as the pistillate parent was highly
fertile with completely normal bivalent formation.
Segregation for
morphological characters and high fertility in the F2 and BC l indicate
the possibility of gene exchange between these species also.
On the basis of crossability relationships, and on the assumption
that cytoplasm which prevents the development of a hybrid embryo is
v
the evolved cytoplasm, while cytoplasm which permits development of a
hybrid embryo is the original type, evolutionary relationships of these
four species are proposed.
Mung bean and rice bean are assumed to have
been derived independently from a common ancestor,
var. soblobata which is a wild species.
possibly~.
radiata
Black gram and adzuki bean are
considered to be later derivatives from mung bean and rice bean, respectively.
Thus, these four species can be divided into two subgroups,
mung bean with black gram and rice bean with adzuki bean, in which the
two species of each group retain enough chromosomal homology to allow
germplasm exchange within the group.
vi
TABLE OF CONTENTS
ABSTRACT • . . •
iii
LIST OF TABLES
ix
LIST OF FIGURES.
xi
INTRODUCTION ••
1
LITERATURE REVIEW
Taxonomy •
3
Cytology
5
Interspecific hybridizations involving the
four Vigna species
5
Embryo abortion. . . .
12
Methods of counteracting hybrid embryo
abortion . . . . . . • . . . . . . . .
13
MATERIALS AND METHODS
Parental lines
15
Methods.
20
RESULTS AND DISCUSSION
A.
B.
Intraspecific Crosses .
23
Crossability • .
23
Meiosis. . •
25
Interspecific Crosses .
25
1.
25
Crosses between adzuki bean and black gram. •
Crossability . .
25
Embryo culture
29
vii
TABLE OF CONTENTS (Continued)
Page
2.
3.
4.
5.
Crosses between black gram and rice bean.
31
Crossabili ty
31
Embryo culture •
33
Morphology of the hybrid
35
Crosses between adzuki bean and mung bean
37
Crossability • •
37
Embryo culture •
39
Morphology of the hybrid •
39
Fertility of the hybrid.
41
Meiosis in the hybrid
43
Crosses between mung bean and rice bean
43
Crossability • •
43
Embr.yo culture •
47
Plant growth and morphology of the hybrids
49
Fertility of the hybrids
53
Meiosis in the hybrids •
53
Amphidiploid .
55
Crosses between black gram and mung bean.
59
Crossability . •
59
Embryo culture •
61
Plant growth and morphology of the hybrids •
61
Fertility of the hybrids
66
Meiosis in the hybrids •
66
The F2 and backcross generations •
68
viii
TABLE OF CONTENTS (Continued)
Page
6.
Crosses between adzuki bean and rice bean
72
Crossability •
72
Embryo culture
74
of the hybrid .
76
Fertility of the hybrid. .
76
Meiosis in the hybrid. . •
80
The F2 and backcross generations •
80
Mo~phology
GENERAL DISCUSSION AND CONCLUSIONS .
84
LITERATURE CITED • • . • • . . . . •
90
ix
LIST OF TABLES
Table
The or~g~ns of nine parental lines of four oriental
bean species..
•••• • • . •.
• •••
16
Characteristics of lines of oriental bean species
used in crosses
..••....• • • • . . .
17
Pod and seed set in pollinations within oriental
bean species . . • . . • • • • . . . • • • • . . .
24
4.
Crossability between adzuki bean and black gram •
28
5.
Results of culturing hybrid embryos from crosses
between adzuki bean and black gram. . . . • •
30
6.
Crossability between black gram and rice bean .
32
7.
Results of culturing hybrid embryos from crosses
between black gram and rice bean • . . . . . • .
34
Morphological comparison of black gram (B2), rice
bean (Rl), and the hybrid between them. . . . • .
36
Crossability between adzuki bean and mung bean.
38
Results of culturing hybrid embryos from crosses
between adzuki bean and mung bean • • . • . . . • •
40
Morphological comparison of mung bean (M3), adzuki
bean (AI), and the,hybrid between them.
. ..•
42
2.
3.
8.
9.
10.
11.
12.
Crossabilit~
13.
Results of culturing hybrid embryos from the
cross between rice bean and mung bean • . .
48
Morphological comparison of mung bean, rice bean,
and the hybrids between them. . • . . . . .
52
14.
15.
between mung bean and rice bean.
Meiotic analysis of hybrids of mung bean X rice
bean. . . . . . . . . . . .
16.
46
. . . . .
Crossability between black gram and mung bean .
54
60
x
LIST OF TABLES (Continued)
Table
Results of culturing hybrid embryos from the
cross between black gram and mung bean. • • •
62
Morphological comparison on mung bean, black
gram, and the hybrids between them. . • • • .
65
Meiotic analysis of hybrids of mung bean X
black gram. . . • • • • • • • . • • • • • •
67
The F2 generation of mung bean X black gram
crosses . .
. . . . . . . . . .
69
Backcrosses of hybrids of mung bean X black
gram to their parents • • • • • • • • • • •
71
22.
Crossability between adzuki bean and rice bean.
73
23.
Results of culturing hybrid embryos from
crosses between adzuki bean and rice bean •
75
Morphological comparison of rice bean (R2),
adzuki bean (A2), and the hybrid between them
79
Backcrosses of the rice bean (R2) X adzuki
bean (A2) hybrid to the parental species
82
Summary of hybridizations among the four
Vigna species . . • • • •
85
17.
18.
19.
20.
21.
24.
25.
26.
xi
LIST OF FIGURES
Figure
1- 4.
Seedlings of parental species.
18
5- 8.
Mature plants of parental species.
18
9.
Chromosome pairing at metaphase I of a
parental species (R2). • . . . • • .
26
The hybrid of black gram (B2) X rice bean
(Rl) obtained by embryo culture. . .
26
The hybrids of mung bean (M3) X adzuki
bean (AI) obtained by embryo culture .
26
Meiosis in mung bean (M3) X adzuki bean
(AI) hybrids • . • .
. . . .
44
19-20.
The hybrids of mung bean X rice bean • •
50
21-23.
Meiosis in mung bean X rice bean hybrids
50
24-25.
Mung bean (M2) X rice bean (R2) amphidiploid
(C2) plants . • • . . • . • • . . . . • . • •
56
Meiosis in the mung bean X rice bean amphidiploids .
•... . • . • • • • . . • •
56
10.
11-12.
13-18.
26-29.
30-31.
The hybrids of mung bean (M2) X black gram
(B2) . . . . . . . . . .
.. . . . . .
63
32-35.
Meiosis in mung bean X black gram hybrids . .
63
36.
A BCl plant from mung bean X (mung bean X
black gram) (M2 X F l of M2 X B2) .
63
The hybrids of rice bean (R2) X adzuki bean
(A2) obtained by embryo culture. . . •
77
Regular meiosis in rice bean (R2) X adzuki bean
(A2) hybrids
• . • .
77
37-38.
39-41.
42-43.
F2 plants of rice bean (R2) X adzuki bean
(A2) . . . . . . . . . . . . . . . .
77
xii
LIST OF FIGURES (Continued)
Figure
44.
Proposed evolutionary relationships of the
four Vigna species • • • . • • • . . • • . .
88
INTRODUCTION
Interspecific hybridization, followed by natural polyploidy, has
played an important role in the evolution of flowering plants in nature
(Stebbins, 1950, 1971; Barber, 1970; Knoblock, 1972).
Experimental interspecific hybridization is used to study the nature
of isolating mechanisms operating between species and to study phylogenetic relationships.
In plant breeding, interspecific hybridization has been used when
desired characters are not found in the range of variation already
present in the species.
Other advantages of interspecific hybridiza-
tion could be the possibility of producing hybrids with higher mutation
rates (Stebbins, 1969), increased environmental adaptiveness (Stebbins,
1969) or combinations of new characters not present in either parental
species (Rick, 1967; Knoblock, 1972; Harlan, 1976).
Therefore, inter-
specific hybridization is a promising tool by which a plant breeder can
create broader genetic diversity.
The oriental species of beans, Vigna radiata (L.) Wilczek (mung
bean),
~.
mungo (L.) Hepper (urd or black gram),
Ohwi and Ohashi (adzuki bean),
and~.
~.
angular is (Willd.)
umbellata (Thunb.) Ohwi and
Ohashi (rice bean), are major pulse crops in a large part of Asia, the
eastern part of Africa, and some other parts of the world.
Although
the taxonomy of this group has undergone many changes, they have always
been treated as a related group and referred to as the 'mung' group or
oriental beans.
Characteristics which distinguish them from other
beans are their Asian origin and their yellow flowers.
2
The purpose of this study is to determine the nature of
reproductive barriers existing between these four species, and to assess the possibility of overcoming the barriers, which would allow the
exploitation of germplasm resources beyond individual species boundaries
in breeding this group of valuable beans.
LITERATURE REVIEW
Taxonomy
The Leguminosae is economically one of the most important groups
of flowering plants, providing a variety of food sources, animal feeds
and raw materials.
Some members are grown as soil building crops or as
ornamentals (Leppick, 1966; Purseglove, 1968; Hutchinson, 1969; Heywood,
1971).
It is a natural group, easily defined taxonomically by a single
character, a special form of fruiting capsule called a 'legume'
(Maekawa, 1955; Tuton, 1958).
There is still no general agreement on the treatment of major
taxa.
Some treatments divided the family Leguminosae into three sub-
families; Caesalpinoideae, Mimosoideae and Lotoideae (syn. Papilionoideae, Faboideae, Papilionatae) (Laurence, 1951).
The Swartzioideae
is sometimes separated from the Caesalpinoideae and appears as a
fourth subfamily (Heywood, 1971).
Hutchinson (1973), however, elevates
the Leguminosae to an order, Leguminales, comprising three separate
families; Caesalpinaceae, Mimosaceae and Fabaceae (syn. Papilionaceae).
The Fabaceae, due to the distinctive floral structure of a
usually gamosepalous calyx and papilionate corolla, is easily distinguished from the members of other subgroups.
The Phaseolineae, a
tribe of Fabaceae, includes all the cultivated beans.
Members be-
longing to this tribe have bearded styles and beaked or spirally
twisted keels, and usually have stipels, inflorescences with swollen
nodes and uniform anthers.
4
There has also been a great deal of confusion in the minor taxa of
oriental beans, chiefly because of the shape of their keels.
The keel
of these beans is curved like that of some members of the genus Vigna
and slightly twisted spirally at the same time.
The spirally coiled
keel is a distinctive characteristic of the genus Phaseolus.
Oriental beans had been generally included in Phaseolus L. until
Verdicourt (1970) revised the taxonomy of this group of beans to put
them into the subgenus Ceratotropis (Piper) Verde. of the genus Vigna
Savio
The Agricultural Research
revision (Gunn, 1973).
Servi~e
of U.S.D.A. recognized this
The basis of Verdicourt's revision is that
these beans have a medifixed stipule, septate pods,and markedly reticulate pollen compared to the basifixed stipules, non-septate pods, and
pollen grains with either no obvious sculpture or only a very fine
reticulation in Phaseolus beans.
In addition to morphological
characters, Verdicourt also relied on free amino acid patterns and
serological reactions which confirm that this group is related more
closely to the other members of Vigna than to Phaseolus.
This group of beans differs from other members of Vigna by having
the stipule distinctly peltate and the style beaked beyond the stigma
with the thickened part strongly curved.
The botanical names revised by Verdicourt are Vigna angularis
(Willd.) Ohwi and Ohashi for adzuki bean,
black gram,
y.
y.
radiata (L.) Wilczek for mung
(Thunb.) Ohwi and Ohashi for rice bean.
be used to alleviate confusion.
mungo (L.) Hepper for
bea~and
y.
umbellata
Hereafter common names will
5
The synonyms frequently appearing in the literature are; Phaseolus
angularis, Dolichos angu1aris and Azukia angularis for adzuki bean;
~.
mungo,
~.
mungo and
radiatus,
~.
radiata and
pubescens,
~.
~
~.
ricciardinus,
mungo for black gram;
aurea for mung bean;
Q.
umbellata,
y.
~. ~, ~.
and~.
calcarata,
calcaratus,
and~.
~.
um-
bellata for rice bean.
Cytology
According to Darlington and Janaki-Ammal (1945), chromosome
numbers for these four species were first reported as 2n
Karpechenko in 1925, but Rau reported 2n
= 24
= 22 by
for black gram in 1929.
Later Sen and Ghosh (1960), Dana (1966a), De and Krishnan (1966a) and
Chaurasia and Sharma (1974) all reported 2n = 22 for black gram.
Kaul
(1970) occasionally observed precocious separation of one bivalent in
meiosis of mung bean that was regular otherwise.
Interspecific hybridizations involving the four Vigna species
The first reported interspecific hybridization among these
species was between mung bean and black gram (Sen and Ghosh, 1960).
Other studies on crosses between mung bean and black gram were conducted by Boling et ale (1961), Khanna et ale (1962), Dana (1966a), De
and Krishnan (l966b), AVRDC (Anonymous, 1974), and Singh and Singh
(1975).
The objective of making this cross was to combine the agro-
nomic and horticultural qualities and early maturity of mung bean
with the high yield potential, high content of sulfur-bearing amino
acids, plant vigor, resistance to many kinds of diseases and insects,
and tolerance to adverse weather conditions of black gram.
6
Partially filled viable seeds were produced only when mung bean
was used as the pistillate parent.
The reciprocal cross failed.
Hybrid
lethality was observed at all stages of development from germination to
flowering.
Lethality, weakness and sterility continued to occur in the
F2 generation.
The F2 and backcross to mung bean produced plants with
combined characters of both parents.
This implies the possibility of
germplasm interchange between these two species.
In the Fl plants the average number of bivalents at metaphase I
was 9 to 10.
Meiotic irregularities such as univalents, quadrivalents,
laggards, improper congregation of the chromosomes on the equatorial
plates and precocious movements of chromosomes were often observed.
Quadrivalents occurred at the frequency of 0.2 per cell, which suggested that a reciprocal translocation occurred (Dana, 1966a).
Based
on pachytene analysis De and Krishnan (1966b) found that a terminal
deletion and an interstitial duplication were also involved.
Hybrid inviability, weakness, sterility and breakdown are considered to be reproductive barriers between these two species (Dana,
1966a).
However, the strength of the isolating mechanisms varies,
depending upon the parental genotypes used in producing the hybrids.
Therefore, mung bean and black gram are considered to be closely
related species which differ chromosomally by one major reciprocal
translocation and several minor modifications.
Dana (1966a) gave the
genomic notation AA for the two species.
Dana (1966b) also reported crossing mung bean and rice bean.
The objective of this cross was to transfer the hypogeal germination
of rice bean into mung bean.
Partially filled viable seeds were
7
produced only in the cross, mung bean X rice bean.
In the reciprocal
cross, young pods dropped in the early stages of development.
Meiosis
of Fl plants was highly irregular withunivalents, bridges, precocious
separation of chromosomes, and improper arrangement of chromosomes on
equatorial plates.
The modal chromosome pairing was 811 + 61.
had only 1.1% stainable pollen and was completely sterile.
The Fl
A colchi-
cine-induced amphidiploid showed gigas characteristics with much increased stainable pollen (70.7%) and many pods with viable seeds.
The
isolating barriers existing between these two species were considered
by Dana to be hybrid inviability and sterility.
The hybrid sterility
was interpreted as chromosomal with a possible genic cause involved.
The genomic notations AA for mung bean and AlAI for rice bean were
proposed.
Hybrids of mung bean X rice bean were also produced by Sawa (1973)
from cultured embryos.
This cross was made for the purpose of trans-
ferring the cyst-nematode resistance of the mung bean into adzuki bean.
The bean which he originally considered to be a semi-wild relative of
adzuki bean, however, was later identified as a local variety of rice
bean (Sawa, 1974).
sterile.
The Fl was very vigorous but completely seed
Abnormal polyspory in microsporogenesis was considered to be
responsible for the hybrid sterility.
Stainable pollen in the colchi-
cine-induced amphidiploids was 58 to 88% compared to 2.5% in the Fl.
The average seed set was 24.5% and 22 bivalents were counted at
metaphase I of the C2 plants.
In their mung bean breeding program in AVRDC (Anonymous, 1974,
1975), mung bean X rice bean hybrids were produced.
The specific
8
objective is to introduce the resistance to diseases and insects and
the high methionine content of the rice bean into mung bean.
The Fl
plants were sterile but produced a single pod with four seeds.
The F2
plants were identified as spontaneous amphidiploids (Baker, personal
communication).
Reciprocal backcrosses to both parental species were
tried without success.
Al-Yasiri and Coyne (1966) classified the cross black gram X rice
bean as a partially compatible cross in which pods collapse in the
early stages of development, and the reciprocal cross as incompatible
in which no pods set.
The only success with a cross between thse two species was reported by Biswas and Dana (1975).
Hybrids were raised by culturing
the embryonic axes of the dried shrivelled seeds produced only in
black gram X rice bean.
Differential compatibility determined by the
female genotypes was noticed.
The hybrid plants were weak and stunted
in growth during the winter but grew vigorously in summer.
In the Fl'
stainable pollen was only 0.7% on the average and no pod set was noticed.
The number of bivalents in the Fl, some of which were loosely
paired, ranged from 3 to 6 in most of the cells observed.
and bridges were occasionally found.
Quadrivalents
Colchicine-induced amphidiploids
produced 81% fertile pollen and mature pods with viable seeds.
Hybrid
inviability due to incompatibility between embryo and endosperm as well
as seedling lethality, hybrid weakness, and hybrid sterility were
considered important reproductive barriers between these two species
by Biswas and Dana (1975).
9
The adzuki bean X rice bean cross is partially compatible, while
rice bean X adzuki bean is incompatible according to the classification
of Al-Yasiri and Coyne (1966).
No oth.er
r~cord
has been found of
successful crosses between these two species, except a brief comment
by Sawa (1973) that fertile hybrids had been obtained by culturing
young embryos from the cross, rice bean X adzuki bean.
There is apparently no record of successful crossing of adzuki
bean with black gram or mung bean.
The cross, mung bean X adzuki bean,
was attempted at AVRDC (Baker, personal communication) without success.
Crosses between adzuki bean and black gram in both directions are rated
as incompatible by Al-Yasiri and Coyne (1966).
An unidentified natural tetraploid species of Vigna, which has
been considered to be of amphidiploid origin (Dana, 1964; Krishnan and
De, 1970) and which most likely is the same species identified as
Vigna radiata (1.) Wilczek var. glabra (Roxb.) Verdc. by a USDA taxonomist (Swindell et al., 1973), has been crossed with mung bean, rice
bean and black gram (Dana, 1964, 1965, 1968; Krishnan and De, 1968a,
b).
A hybrid from the cross, mung bean X the natural tetraploid
species, was obtained by Dana (1965).
The hybrid set pods which shed
within 2 to 3 days and the Fl plant died 14 days after flowering began.
Apparently well-developed pods harvested from the reciprocal cross
contained only shrivelled nonviable seeds.
Krishnan and De (1968a) crossed a colchicine induced autotetraploid mung bean with the natural tetraploid species.
Hybrids were
obtained only when the auto tetraploid mung bean was used as the female.
10
The hybrids were completely seed sterile.
The average chromosome
pairing in the Fl plants was 1511 + 141'
Black gram was crossed with the tetraploid species by Dana (1968).
Completely sterile hybrids with 0.83% stainable pollen were obtained
only when black gram was used as the female parent.
Colchicine induced
amphidiploids (allohexaploids) had gigas characteristics, were semifertile, and produced a few small pods with viable seeds.
Krishnan and De (1968b) obtained completely sterile hybrids when
they crossed a colchicine induced autotetraploid black gram with the
tetraploid species.
The average chromosome pairing in the Fl was
0.021V + 0.41111 + 7.7711 + 16. 15 1'
Dana (1964) obtained hybrids from the cross of the tetraploid
species X rice bean by culturing young ovules.
Chromosome pairing in
the completely sterile hybrids was 1111 + 111 in most of the cells
examined.
Thus, he concluded that the natural tetraploid species is an
amphidiploid with one genome homologous with the genome of rice bean.
Extensive studies were conducted on a cross between mung bean and
V. trilobata by Dana (1966c,d) and Dana and Das (1974).
V. trilobata
(syn. Dolchos trilobatus, Phaseolus trilobatus and!. trilobus) is a
forage legume.
the female.
Hybrids were obtained only when mung bean was used as
The hybrids were partially fertile with 31% stainable pol-
len on the average.
of the cells studied.
In the hybrids 11 bivalents were counted in 39.9%
Meiotic irregularities, such as quadrivalents,
precocious separation of chromosomes, failure of chromosomes to congregate on the equatorial plate, bridges, and laggards were often noticed.
11
Highly fertile amphidiploids were induced artificially from this
hybrid by colchicine treatment.
Natural amphidiploids were also raised
from seeds which set on one fertile branch, which probably originated
from somatic chromosome doubling in the meristem of a lateral bud.
Natural amphidiploids and triploids were also found among the F2
progeny of diploid hybrids.
Restitution during gametogenesis was
thought to be responsible for the production of these polyploids from
the diploid hybrid.
Meiosis in the amphidiploids was fairly regular
with 22 bivalents in most of the cells examined.
The same genomic notation of AA was given to both of these species.
Hybrid inviability, weakness, and sterility were considered as the main
isolating barriers acting between these two species.
Strand (1943) reported that he obtained fertile progenies from
the cross Phaseolus vulgaris X black gram.
Kloz et ale (1960), however,
questioned this result on the basis of their distant relationship in
serological reaction.
A positive relationship between the degree of
similarity in protein characters and crossability, graft affinity,and
geographical origins was postulated.
According to Yarnell (1965) ,
Strand in 1950 produced a hybrid seedling from the cross, Phaseolus
vulgaris X adzuki bean.
later turned white.
The seedling was pale green at first and
Due to the commonly accepted distant relationship
between black gram and adzuki bean and!. vulgaris, and the difficulty
in crossing even closely related species, these last two reports, especially the former one, are very questionable.
12
Embryo abortion
Hybrid inviability is a common phenomenon in interspecific
crosses.
Embryo abortion is a type of inviability which operates in
the early developmental stages of zygotes.
Somatoplastic sterility, in which retarded embryo growth, hyperplasia of the nucel1us, and failure of normal development of the conducting elements are considered to be responsible for ovule collapse,
was postulated as the cause of hybrid seed failure in species crosses
in Nicotiana by Cooper and Brink (1940) and Brink and Cooper (1941).
Satina et al. (1950) felt that the formation of ovular tumors in hybrid
ovules caused embryo abortion in incompatible species crosses of
Datura.
In an extensive review on plant genetic tumors, Smith (1972)
concluded that ovular tumors in interspecific hybrids which are associated with the failure of hybrid embryos are genetical and the
result of the interaction between the different parental contributions.
The tumor-prone hybrid plants synthesize
~r
accumulate greater than
regulatory amounts of growth-promoting substances such as auxins and
cytokinins.
Hybrid embryo abortion is known to occur widely in the legumes.
Johansen and Smith (1956) reported that the cause of embryo abortion
in the cross, Arachis hyposia X A. diogoi, is due to slow embryo
growth, malfunctioning of the endosperm, and failure of endosperm
differentiation.
Griton and Wierzbica (1975) presumed that the failure
of endosperm to develop and function normally leads to embryo starvation in the intergeneric cross of Pisum sativum :. Vicia faba.
Polytene
chromosomes in the suspensor of Phaseo1us vulgaris were discovered by
13
Nag1 (1969), which indicates that the suspensor is an area of high
metabolic activity.
Haq et al. (1973) assumed that the low level of
activity of the suspensor region, as shown by low staining ability, has
some connection with the retarded development of the endosperm and embryo in the cross, Phaseolus coccineus X
f.
vulgaris.
Furuya and Soma
(1957) observed that a high concentration of auxins brought about
suppression of growth and proliferation of tumorous tissue in the culture
of P. vulgaris embryos.
Methods of counteracting hybrid embryo abortion
Skirm (1942) regarded embryo culture as a promising tool in plant
breeding, by which interspecific seed sterility expressed by the abortion
of partially developed embryos can be overcome.
According to Blakeslee
(1945), embryos at or later than the torpedo stage can be excised and
cultured.
Young embryos from Phaseo1us vulgaris X f. acutifolius, which
otherwise abort, were successfully cultured in White's medium by
Honma (1955).
The older and larger embryos grew faster and sturdier.
He also found that a high sucrose concentration in the medium favored
root growth while a low concentration favored shoot growth.
Al-Yasiri
and Coyne (1964) reported that the application of naphthalene acetamide
alone or together with potassium gibberellate to the stigma just before
pollination delayed embryo abortion in the cross, Phaseolus vulgaris X
P. acutifolius, so that the resulting embryos were large enough to be
cultured in vitro.
Ibrahim and Coyne (1975) reported that application of White's nutrient solution to the stigmatic surface prior to pollination enhanced
14
pod set, while detachment of pods from plants prevented embryo abortion
in the cross Phaseolus coccineus X K. vulgaris.
Pods showing signs of
embryo abortion were left on the plant but partially detached by breaking
the pedicels, or taken off from the plant and kept in an air-tight
plastic bag in a growth chamber.
into viable seeds.
Under these treatments, embryos grew
They considered that inhibiting substances produced
in P. coccineus leaves and translocated to the pods were the cause of
embryo abortion.
Baker et al. (1973) reported that the immunosuppressant, amino
caproic acid, promoted pod set and development in reciprocal crosses
between mung bean and rice bean.
Embryo culture has also been employed to produce interspecific
hybrids between species belonging to the mung group.
Dana (1964) pro-
duced hybrids of the tetraploid species X rice bean by culturing young
embryos.
Sawa (1973) followed Honma's procedure to obtain hybrids from
crosses of mung bean X rice bean and rice bean X adzuki bean.
Biswas
and Dana (1975) obtained hybrids from a black gram X rice bean cross
only by culturing the poorly developed embryonic axes dissected out of
the water-soaked seeds.
MATERIALS AND METHODS
Parental lines
Three lines of mung bean and two lines each of adzuki bean, black
gram, and rice bean were used as parents for this study.
The origins
and some morphological characteristics are shown in Tables 1, 2 and
Figures 1-8.
Hereafter, the lines will be referred to by the symbols
listed in Table 1.
All lines were received from the Western Regional
Plant Introduction Station at Pullman, Washington and increased.
Characters such as germination habit, petiole length of primary
leaves, and hairiness on the pod clearly divide these species into two
groups, mung bean and black gram versus adzuki bean and rice bean.
Black gram and mung bean have epigeal germination, sessile primary
leaves, and hairy pods while adzuki bean and rice bean have hypogeal
germination, long-petioled primary leaves, and glabrous pods.
Black gram can be easily distinguished from mung bean because
black gram has upright pods at maturity, thick and smooth pods, and a
concave hilum cushion in contrast to radiate or drooping pods,
slightly constricted pods, and straight hilum in mung bean.
Two of the
three mung bean lines and both of the black gram lines were slightly
delayed in flowering in the summer.
Summer flowering was sparse in
black gram, but not in mung bean.
Adzuki bean can be distinguished from rice bean by broadly cordate
primary leaves, conspicuously constricted pods, and a straight hilum,
in contrast to lanceolate primary leaves, smooth pods, and a concave
hilum cushion in rice bean.
The photoperiodic response seems to be
16
Table 1.
The origins of nine parental lines
of four oriental bean species
Common names
P. 1. No.
Symbols
Adzuki bean
175240
Al
India
237689
A2
Japan
174907
Bl
India
208462
B2
Nepal
200840
Ml
Burma
207504
M2
Afghanistan
377231
M3
India
276369
Rl
India
322571
R2
Brazil
Black gram
Mung bean
Rice bean
Origins
Table 2.
Characteristics of lines of oriental bean species usee in crosses
Characters
---
Lin e
Al
Germination
---'
Primary leaves
not concave
smooth
!
upright
slight
smooth
concave~t concave
~
..yellow
maroon
Pod length (cm)
-Seed size (mm):
length
width
6.1
7.5
8.3
6.2
5.1
5.5
4.2
7.7
4.8
Seed weight (mg)
11.4
6.6
Days to flower:
winter
summer
32
30
32
Seed number per pod
3.8
5.7
B2
lanceo1ate
radiate or drooping
yellow,
purple
sped:1.
33
BI
hairy
---
drooping
HilUI:l cushion
-
M3
sessile
conspicuous
---
m
long-petiolate
Constriction of pod
Seed color
IU
epigea1
broadly cordate
Pods at maturity
fl
hypogea1
glabrous
Pod
Shape of primary leaves
R2
R1
A2
dark grey and
white spotted
grey-
ye110w-
7.2
6.4
7.5
4.4
4.0
7.1
4.1
4.0
3.4
3.8
3.5
4.3
3.8
5.2
3.9
4.8
4.3
11.2
'1.5
4.1
4.3
5.0
4.8
5.4
45
no f1.
41
45
43
49
31
36
30
32
30
37
37
43
6.3
11.1
10.2
11.1
4.7
5.2
red
6.4
8.4
.
concave
green
green
green
t-'
......
18
Figs.
1-4. Seedlings of parental species: 1) adzuki bean (A2);
2) black gram (Bl); 3) mung bean (M2); 4) rice bean (Rl).
Figs.
5-8. Mature plants of parental species: 5) adzuki bean (A2);
6) black gram (B2); 7) mung bean (M2); 8) rice bean (R2).
20
day-neutral in both adzuki bean lines and in one rice bean line (R2),
but the other rice bean line (Rl) requires short days to flower.
Plant Inventory number 322571 is listed as adzuki bean.
However,
the morphological characters (Table 2) as well as crossability with
other species (Tables 3, 6, 12 and 22), clearly show that this line is
rice bean.
This line has 1anceolate primary leaves, a smooth pod, and
a concave hilum cushion which are specific characteristics of rice
bean and used to distinguish rice bean from adzuki bean.
Moreover,
P.I. 320571 crosses readily with the other line of rice bean, Rl
(Table 3), and fails to produce any mature seeds in crosses with the
two lines of adzuki bean, Al and A2 (Table 22).
Therefore P.I. 322571
is treated as a line of rice bean, with the symbol R2.
Methods
Cultural conditions:
One to three plants were grown in each 20 cm
plastic pot in a mixture of 3 parts soil and 1 part vermiculite.
Three
to 7 grams of Osmocote (14-14-14) was applied three times at 2 week
intervals from the time of germination.
Plantings of parental lines
were made at 2 week intervals in order to have flowers available whenever desired.
Crossing methods:
All crosses were made in the greenhouse from
9 to 11 a.m. during the winter.
A modification of the mung bean
hybridization technique of Boling et al. (1961) was used.
Plump buds
due to open on the next day were selected as female flowers.
These
flower buds were opened with sharp-pointed forceps along the suture
of the standard, and the standard and wings were pulled down.
After
that, one-third of the keel was taken away, the pistil and stamens
21
were exposed, and the ten stamens were removed.
The pistil was
pollinated immediately by rubbing the stigma with the pollen-laden
pistil from an open flower of the desired male parent.
After pollina-
tion, the wings and standard were replaced in their original position
to protect the pollinated pistil from drying out.
The walks and benches of the greenhouse were wet with water before
and after the crossing operation in order to keep the humidity high.
Pod setting was recorded 1 week after pollination.
Germination of hybrid seeds:
Hybrid seeds were germinated in
Jiffy-7 pots, and the seedlings were transplanted in 1 to 2 weeks to
plastic pots with the soil mixture.
Embryo culture:
Interpsecific hybrid embryos at various developing
stages were dissected out and cultured in Vacin and Went medium according to the procedure reported by Sagawa and Va1mayor (1966).
Seedlings obtained by embryo culture were transplanted to Jiffy-7
pots from the test tubes when the shoot
~as
at least 2 cm long.
After
2 to 5 days under shade, they were moved to the greenhouse and
gradually exposed to direct sunlight by decreasing the saran cloth
covering over a week-long hardening period.
They were then trans-
planted to plastic pots with the soil mixture.
Pollen fertility:
Pollen fertility was estimated from the per-
centage of pollen grains that were stained uniformly by 0.5% cotton
blue in 1actopheno1.
At least 1,500 pollen grains from five flowers
were counted for each plant.
Cytology:
For cytological observations, flower buds were collected
from 5 to 7 p.m. and fixed in a modified Carnoy's fixative of 6 parts
22
chloroform, 3 parts absolute ethanol and 1 part glacial acetic acid, as
used by Carr (1976).
Anthers from the buds kept in the fixative for
more than 1 week, were squashed in 1% acetocarmine and mounted with
Hoyer's mounting medium following the Beeks' Permanent Squash Method
(1955).
Colchicine treatment:
An attempt to induce amphidiploidy was
made by treating the cuttings with colchicine according to Smartt and
Haq (1972).
Vigorous branches taken from the hybrid plants were
treated with colchicine by immersing the cut ends into 0.1% aqueous
solution for 6 hours in a shady place.
The cuttings were then washed
and rooted under a mist propagator for 2 to 3 weeks.
RESULTS AND DISCUSSION
A.
Intraspecific Crosses
Crossability
Self and cross pollinations of lines within species were made
after emasculation to determine the fertility of female and male gametes
and the effectiveness of the crossing technique as well as the seed
development following fertilization (Table 3).
There was a highly significant difference in pod set between
species, but the differences between female and male parents or between self and cross pollinations within species were not significant
except for the two rice bean lines.
R2, used as a seed parent, had a
higher percentage of pod set (68.8%) than RI (26.7%) but there was no
difference between these two lines as pollen parents.
The difference in pod set between species and between two lines
of rice bean is probably due to differences in tolerance to floral
injury from emasculation and pollination.
The extremely low pod set
in adzuki bean is at least partly due to scant pollen production during
the winter.
Pods that set also reached maturity, except in Al X Al, Al X A2,
and R1 X R2 in which one pod in each combination dropped in the early
stage of development.
Ripe pods harvested from artificial pollinations
contained normal seeds, but the seed number per pod was slightly lower
than in open-pollinated flowers (cf. Tables 2 and 3).
The results imply that female and male reproductive systems function normally and that there areno post-fertilization obstacles, such
24
Table 3.
No. of
flowers
pollinated
No. of
pods
harvested
%
pod set
No. of
seeds
obtained
No. of
seeds
per pod
Al
A2
Al
A2
23
23
20
21
87
4
6
5
5
20
17.4
26.1
25.0
23.8
23.0
9
13
13
15
50
2.3
2.2
2.6
3.0
2.5
B1
B2
B1
B2
22
26
20
20
88
9
7
6
6
28
40.9
26.9
30.0
30.0
31.8
39
29
29
31
128
4.3
4.1
4.8
5.2
4.6
M1
M2
M3
M1
M2
M3
M1
M2
M3
19
20
21
20
19
20
20
23
22
184
13
12
15
15
9
13
7
16
13
113
68.4
60.0
71.4
75.0
47.4
65.0
35.0
69.6
59.1
61.4
119
107
148
116
86
120
71
156
131
1,054
9.2
9.2
9.9
7.7
9.6
9.2
10.1
9.8
10.1
9.3
R1
R2
R1
R2
30
30
32
22
114
11
5
19
16
51
36.7
16.7
59.4
72.7
44.7
52
33
106
78
269
4.7
6.6
5.6
4.9
5.3
Parents
F
Al
A2
M
Total
B1
B2
Total
M1
M2
M3
Total
R1
R2
Pod and seed set in pollinations within
oriental bean species
Total
X2- test for pod set
Comparison
Self vs. cross
pollination
Within species
Adzuki bean
Black gram
Mung bean
Rice bean
0.57 ns
0.87 ns
0.17 ns
39.70**
Between species
20.80**
**significant at 1% level; ns
Between lines
as receptor as pollinator
0.15 ns
0.18 ns
1.07ns
15.90**
= not significant.
0.36 ns
0.870S
0.34ns
o. nns
25
as embryo abortion, to normal seed development in self-pollinations or
interline crosses within the species.
Ten flowers of each of the nine parental lines were left emasculated
without pollination to study the possibility of parthenocarpic fruit development.
Apparently, parthenogenesis does not occur in these species
since fruit develoment was not observed from flowers left emasculated.
Meiosis
All of the parer tal species and interline hybrids within species
regularly formed 11 bivalents at first meiotic metaphase followed by
normal microsporogenesis.
A lack of synchronization in anaphasic
chromosome disjunction, with precocious separation of one to two pairs
of chromosomes was a common phenomenon in these species (Fig. 9).
B.
1.
Interspecific Crosses
Crosses between adzuki bean and black gram
Crossability
In crosses of adzuki bean X black gram, there was a singificant
difference in pod set between the two lines of adzuki bean.
On Al
9 pods out of 70 (12.9%) set, while on A2 only 2 out of 76 (2.7%) set
(Table 4).
The difference between the two black gram lines used as
pollen parents was not significant.
after pollination.
All the pods shed within 12 days
Pod growth was much slower than in open-pollinated
ones and barely reached 2.5 cm in length before shedding.
Loss of
turgidity and yellowing indicated the beginning of pod abortion.
The
embryos at the time of pod abortion were large enough to recognize and
remove.
26
Fig.
9. Chromosome pa1r1ng at metaphase I of a parental species
(R2), showing 11 bivalents with precocious separation of one
bivalent Xl,200.
Fig. 10. The hybrid of black gram (B2) X rice bean (Rl) obtained by
embryo culture.
Figs. 11-12. Fl hybrids of mung bean (M3) X adzuki bean (AI) obtained by embryo culture: 11) seedlings; 12) mature plant.
f
I
9
.,1,. ,
28
Table 4.
Crossability between adzuki bean and black gram
No. of
flowers
crossed
Parents
Female Male
A1
33
37
70
5
4
9
15.2
10.8
12.9
B1
B2
39
37
76
0
2
2
0.0
5.4
2.6
A1
A2
44
35
79
5
9
14
11.4
25.7
17.7
A1
A2
28
30
58
5
6
17.9
20.0
19.0
Total
B1
Total
B2
%
pod set
B1
B2
Total
A2
No.
of
pods set
Total
11
X2_ test for pod set
Comparison
Between A X Band B X A
X2 value
4.46*
Within A X B
16.53**
between A
between B
6.76**
0.09ns
Within B X A
between B
5.63ns
0.04ns
between A
2.2Sns
X2 value significant at 5% (*) or 1% (**); ns = not significant.
29
The crosses of black gram X adzuki bean showed no differences in
pod set between the two lines of black gram or between the two adzuki
bean lines used as pollen parents.
Pod growth was slightly retarded
and abortion usually occurred between 2 and 4 weeks.
Embryos at the
time of pod abortion were fairly large but slightly deformed.
A difference in crossability according to the direction of hybridization is clearly shown in crosses between these two species.
A
higher per cent pod set (18.3%) was found when black gram was used as
the female than when adzuki bean was (7.5%).
Pod set on adzuki bean
was markedly lower than in intraspecific pollinations.
also set fewer pods.
Black gram
However, the scant pollen of adzuki bean might
be responsible for this low set.
gram was used as the female.
Pod abortion was delayed when black
Pods sometimes lasted almost to the
mature stage, while pods aborted within 2 weeks when adzuki bean was
used as the female.
Embryo culture
From three embryos from a 9-day old green pod of an Al X BI cross,
two etiolated seedlings developed which then died.
formed only roots and callus.
The other embryo
Three embryos from a 10-day old dropped
pod of the cross A2 X B2 failed to show any sign of growth (Table 5).
Out of 16 embryos from the cross BI X A2 cultured at various ages
between 13 to 26 days, seven grew into normal-appearing seedlings, two
showed poor embryonic growth with callus, two formed black callus, and
five did not grow at all (Table 5).
The green, normal-appearing
seedlings started to show signs of wilting I to 3 weeks after they were
30
Table 5.
Male
Age of
embryo
(days)
Al
B1
9
A2
B2
Bl
Bl
Parents
Female
B2
Results of culturing hybrid embryos from
crosses between adzuki bean and black gram
State
of
pod
No.
of
embryos
Results
green
3
2 etiolated seedlings
1 only roots and
callus
10
dropped
3
no growth
Al
16
green
1
no growth
A2
13
green
6
2 black callus
4 no growth
14
15
16
green
green
green
1
3
3
seedling died
3 seedlings died
2 poor embryonic growth
with callus
1 no growth
26
dried
3
1 3 p1ant1ets attached,
died
2 seedlings died
10
14
green
green
1
1
no growth
no growth
A2
31
transplanted to soil pots and eventually died.
The hybridity of the
seedlings was evident from the short petioled ovate primary leaves.
The root systems were found to be poorly developed.
A2, two embryos were cultured, one
io
In the cross B2 X
days old and the other 14 days
old; neither showed any sign of growth.
Poor development of the root systems was suspected to be a primary
cause of the seedling mortality, since the seedlings from the cross
Al X B2 showed no other symptoms beside poor root development.
McLean (1946) successfully secured hybrid seedlings containing
shoots but no roots from Datura species crosses by grafting them on
young vigorous plants of a pure species.
Honma (1955) reported that a
high sucrose concentration in the embryo culture medium favored root
growth while a low concentration favored top growth in the culture of
hybrid embryos from the species cross Phaseolus vulgaris X P. acutifolius.
Survival of these hybrids could possibly be attained by
modifying the culture media so as to encourage better root growth or
by grafting them on seedlings of either parental species.
Because of the failure of hybrids between adzuki bean and black
gram to grow, these two species are assumed to be the most distantly
related of the four species considered herein.
2.
Crosses between black gram and rice bean
Crossability
When black gram was used as the female, 69 pods (43.1%) set from
160 flowers pollinated (Table 6).
and reached maturity.
The hybrid pods developed normally
The ripe pods contained two kinds of seeds;
32
Table 6.
Parents
Female Male
Bl
Total
No. of
pods
harvested
No. of seeds
obtained
Empty Filled
22
12
34
50.0
30.8
41.0
14
12
26
0
8
8
43
32
75
Rl
R2
40
37
77
25
10
35
62.5
27.0
45.5
16
8
24
5
9
14
48
17
65
Bl
B2
45
45
90
2
2
4
4.4
4.4
4.4
Bl
B2
40
45
85
0
0
0
Total
R2
Pod set
No.
%
44
39
83
Total
Rl
No. of
flowers
crossed
Rl
R2
Total
B2
Crossability between black gram and rice bean
All of the pods shed
within 3 weeks.
X2_ test for pod set
Comparison
X2 value
B X R vs. R X B
36.73**
Within B X
between
between
Within R X
between
between
19.57**
0.23ns
8.59**
8.88*
4.44*
0.004ns
R
B
R
B
R
B
X2 value significant at 5% (*) or 1% (**); ns
= not
significant.
33
small, undeveloped ones and full-size ones which were slightly crinkled.
When the slightly crinkled seeds were soaked in water, they appeared
normal but the cotyledons were highly distorted and the embryonic axes
were poorly developed.
Neither kind of seed germinated.
When rice bean was used as female, only 4 pods out of 175 (2.3%)
set.
Pods grew very slowly and wilted 3 weeks after pollination.
In addition to the reciprocal difference in crossability between
tr1ese two species, a difference between rice bean lines on pod set was
clearly shown.
Pod set on black gram was normal when pollinated by R2
but significantly decreased when pollinated by Rl.
Rl also failed to
set any pods when pollinated by black gram pollen while R2 set a few
pods.
Embryo culture
From the crosses using black gram as female, 34 hybrid embryos
were cultured.
and 20
f~om
Fourteen embryos were taken from 10- to l4-day old pods
mature pods.
Nine apparently good seedlings were obtained;
3 from young pods and 6 from mature pods in the cross B2 X Rl.
embryo culture results are listed in Table 7.
The
Of the nine apparently
good seedlings, seven died during early seedling stages and only two
very weak dwarf plants survived with arrested growth for more than 3
months, and then died before flowering (Fig. 10).
From crosses using rice bean as female, one embryo from an 8-day
old green pod and five embryos from a l6-day old wilting pod were
cultured.
Of these, three embryos formed callus without embryonic
development and the other three embryos did not grow at all.
34
Table 7.
Parents
Female Male
Bl
B2
Rl
Rl
Rl
B2
Results of culturing hybrid embryos from crosses
between black gram and rice bean
Age of
embryo
(days)
State
of
pod
No.
of
embryos
10
green
3
22
ripening
2
28
ripe
3
15
green
6
18
green
5
19
ripening
1
20
ripening
3
21
ripening
4
24
26
27
ripe
ripe
ripe
2
2
3
8
16
green
wilting
1
5
Results
1 deformed leaves and
callus
2 callus
1 seedling with shoot
tip highly coiled
1 seedling died
1 primary leaves only
2 no growth
1
2
3
3
died after 3 months
seedlings died
no growth
only shoots with
callus
2 no growth
no growth
1 seedling died
2 callus
1 etiolated shoot,
poor roots and
callus
1 seedling died
1 only hypocotyl
elongated
1 no growth
no growth
no growth
2 seedlings died
1 died after 3 months
no growth
3 callus
2 no growth
35
Therefore, embryo culture might be a tool for circumventing the
barriers between these two species of embryo abortion and germination
failure only when black gram is used as the female parent.
weakness and inviability are also strong barriers.
plants with stunted growth never became vigorous.
Hybrid
The extremely weak
It is possible that
the death of the hybrid plants from this particular cross could be
prevented by growing them under a more favorable cultural environment.
Biswas and Dana (1975) reported that they produced hybrids by
culturing embryonic axes of mature seeds from a certain black gram X
rice bean cross.
The hybrid plants, which were weak and stunted in
growth during the winter became vigorous in the summer.
Amphidiploids
induced by chromosome doubling of the completely sterile hybrids produced a few pods with mature seeds.
Therefore, growing the hybrid
plants under long day conditions to encourage vegetative growth, and
trying other parental lines in hybridization might be effective methods
to secure hybrid plants that reach the flowering stage.
The reciprocal cross, rice bean X black gram, is not likely to be
successful because of a strong barrier to fertilization as well as
complete embryo abortion.
Morphology of the hybrid
The hybrid origin of the seedling obtained by embryo culture was
confirmed by their germination habit and the petiole length of the
primary leaves (Table 8).
Germination is epigeal in black gram and
hypogeal in rice bean (See Figs. 2 and 3, and Table 2).
The cotyledons
of the Fl seedlings remained at or a little above the soil surface
without further elongation of the hypocotyl, intermediate between the
36
Table 8.
Characters
Morphological comparison of black gram (B2),
rice bean (Rl), and the hybrid between them
Black gram
(female)
Hybrid
Rice bean
(male)
Germination habit
epigeal
intermediate
hypogeal
Petiole length
of primary
leaves
sessile
short
long
Petiole color
purple
light purple
green
Margin of leaflets
entire
entire
often lobed
37
parents.
The primary leaves are sessile in black gram and
long-peti.olate in rice bean.
The primary leaves of the Fl seedlings
had short petioles, again intermediate between the parents.
The
petiole color in B2 is purple and green in Rl, while that of the Fl
hybrids was intermediate or light purple.
The margin of the trifoliate
leaflets was entire in the Fl hybrids, similar to black gram, not
lobed as in rice bean.
3.
Crosses between adzuki bean and mung bean
Crossability
When adzuki bean was used as the female parent, there were significant differences in pod set between adzuki bean lines (Table 9).
Al
set 33 pods out of 105 (31.4%) and A2 set only 7 pods out of 105 (6.7%).
Differences between the mung bean pollen parents were not significant.
Pod growth ceased in approximately 10 days when pod length reached
1.5 - 2.0 cm.
Pods remained green for as much as 3 weeks longer
without further development.
Embryos barely reached the heart shape
stage before they started to degenerate.
When mung bean was used as the pistillate parent, pod set was
significantly lower on one line of mung bean.
Pod set was only 11.3%
on Ml as compared to approximately 30% on M2 and M3.
Differences be-
tween the adzuki bean pollen parents were not significant.
Pods grew
slowly and usually shed in 1 to 3 weeks after pollination.
Embryos
older than 1 week were fairly large and appeared normal.
No reciprocal difference in pod set was found in these crosses.
However, pod set on mung bean was less than the pod set from
38
Table 9.
Crossability between adzuki bean and mung bean
No. of
flowers
crossed
Parents
Female Male
A1
36
34
35
105
12
10
33
30.6
35.3
28.6
31.4
Ml
M2
43
31
31
105
2
1
4
7
4.7
3.2
12.9
6.7
Al
A2
30
50
80
4
5
9
13.3
10.0
11.3
Al
A2
34
34
68
8
13
21
23.5
38.2
30.9
A1
A2
33
30
63
8
11
24.2
36.7
30.2
M3
Total
M1
Total
M2
Total
M3
%
pod set
M1
M2
M3
Total
A2
No.
of
pods set
Total
11
19
X2- test for pod set
Comparison
A X M vs. M X A
Within A X M
between A
between M
Within M X A
between M
between A
X2 value
0.4l ns
51.16**
16.09**
0.64ns
27.65**
10.28**
0.50*
X2 value significant at 5% (*) or 1% (**); ns = not significant.
39
intraspecific pollinations while pod set on adzuki bean was similar in
both kinds of pollinations.
Clear reciprocal differences were found in
pod growth, embryo development, and the time of embryo abortion on the
two parental species.
Embryo culture
From adzuki bean pods 11 embryos or whole ovules 7 to 21 days old
were cultured (Table 10).
Of these, only one embryo developed into an
etiolated seedling which soon died and another embryo formed only callus
while nine embryos showed no growth at all.
Embryos or ovules older
than 10 days did not show any sign of growth.
Therefore, embryo abor-
tion must precede the suspension of pod growth which usually occurs
around 10 days after pollination.
From mung bean pods, 26 embryos 9 to 19 days old were cultured
and 12 seedlings were obtained, of which 2 were etiolated and soon
died and the rest were apparently normal.
During the early stages
eight seedlings with poor roots died, but two hybrid plants reached
flowering stage.
These two hybrid plants were cultured from 19-day old embryos
taken from a wilting pod of the cross M3 X Al.
Thus, embryo abortion
in the mung bean X adzuki bean cross is apparently delayed until the
pod collapses, and hybrids can be secured by culturing the young embryos from some parental line combinations.
Morphology of the hybrid
The hybrids showed an intermediate germination habit, the epicotyl color of the male parent, and intermediate petiole length, and
40
Table 10.
Parents
Results of culturing hybrid embryos from crosses
between adzuki bean and mung bean
Age of
embryo
(days)
State
of
pod
No.
of
embryos
Results
green
green
dropped
green
1
1
2
1
etiolated seedling
no growth
no growth
no growth
Female
Male
Al
Ml
7
10
11
16
Al
M2
8
green
2
11
13
green
green
1
2
1 callus
1 no growth
no growth
no growth
Al
M3
21
wilting
1
no growth
M1
A1
11
dropped
8
no growth
M1
A2
12
17
green
wilting
6
4
6 seedlings died
2 etiolated
seedlings
2 no growth
M2
A2
12
dropped
1
no growth
M3
A1
19
wilting
2
2 mature plants
M3
A2
9
green
5
2 seedlings died
3 no growth
41
shape of the primary leaves (Fig. 11).
Germination is epigeal in mung
bean and hypogeal in adzuki bean while the cotyledons of the hybrids
rise slightly above the soil surface without further elongation of the
hypocotyl.
The epicotyl color was purple in M3 but light green in the
Fl, resembling the paternal parent, adzuki bean.
The primary leaves
are sessile and lanceolate in mung bean, but long petiolate and
broadly cordate in adzuki bean.
The hybrids had ovate and short
petioled primary leaves.
The leaf color of the hybrid was dark green and the young pods
were hairy, both similar to mung bean, while the trifoliate leaflet
margins were lobed as in adzuki bean (Table 11 and Fig. 12).
Fertility of the hybrid
In the FI hybrids all pods set from open-pollinated flowers and
from controlled selfings were shed in 10 days.
Backcrosses of 20
flowers each of all four possible combinations were made.
Backcrosses
where parental species were used as the females failed to set any
pods.
However, in the backcrosses with the Fl as the female parent
a few pods set, which dropped within 2 weeks after pollination.
Hybrid
sterility, which is complete, is considered to be an absolute barrier
to hybridization between these two species, at least at the diploid
level.
Induction of juvenile shoots, which could be used for vegetative
propagation or colchicine treatment, was attempted by applying fertilizer, long-day treatment and cutting back of the main stems.
of the treatments were effective and the plants died.
None
42
Table 11.
Morphological comparison of mung bean (M3), adzuki
bean (Ai), and the hybrid between them
Characters
Mung bean
(female)
Hybrid
Adzuki bean
(male)
Germination habit
epigea1
intermediate
hypogeal
Color of epicoty1
base purple
light green
light green
Petiole length
of primary
leaves
sessile
short
long
Shape 'Jf primary
leaves
1anceo1ate
ovate
broadly
cordate
Color of leaves
dark green
dark green
green
Margin of leaflets
entire
often lobed
often lobed
Young pod
hairy
hairy
glabrous
43
Meiosis in the hybrid
Chromosome pairing at metaphase I in the hybrids was extremely low.
The pairing ranged from 0 to 4 pairs with a mean of 2.3911 + 17.221 and
a mode of 211 + 181 in 28 cells examined.
The maximum and modal pair-
ings are illustrated in Figs. 13 and 14.
Chromosomes in metaphase II were scattered randomly throughout the
P.M.C. (Fig. 15) instead of in an orderly arrangement on the two
equatorial plates.
In telophase, adjacent chromosomes congregated to
form microspores at random (Figs. 16 and 17).
Examination of 54 sporads
showed 66.7% (36) diads (Fig. 18) and 5.6% (3) diads with one or two
smaller microspores.
Only 7.1% (4) were tetrads while 19.6% (11) were
monads or monads with one or two micronuclei.
Extremely low chromosome pairing due to lack of homology, followed by abnormal successive divisions, is considered to be responsible
for the hybrid sterility.
Because of the sporadic arrangement of the
chromosomes instead of orderly congregation on the equatorial plates
at second metaphase, sporocytes which form from diads do not seem to
be restitution gametes, which would contain whole sets of chromosomes
of both genomes of the two species.
4.
Crosses between mung bean and rice bean
Crossability
This cross had a much higher success rate when mung bean was used
as the female (Table 12).
5.6% on rice bean.
Pod set was 53.1% on mung bean but only
Seeds were produced only on mung bean.
44
Figs. 13-18. Meiosis in mung bean (M3) X adzuki bean (A1) hybrids:
13) maximum pairing configuration of 411 + 141 (biva1ents are
indicated by arrows); 14) modal pairing of 211 + 181; 15) metaphase II showing 22 chromosomes scattered randomly; 16) congre~
gation of chromosomes into two groups at anaphase II;
17) telophase PMC forming a two-pole cell; 18) three diads.
Figs. 13-17 X1,200 and Fig. 18 X480.
•
•
'.
~
• •
• •
•
••
13
14
,"
... .1
.......'"
"
;
~"
'
.
...
16
•
•
•
...
,
•
I'"
•"'.
••
46
Table 12.
Parents
Female Male
M1
No. of
flowers
pollinated
Total
M3
Empty
Filled
Seeds obtained
16
22
38
48.5
62.9
55.9
15
17
32
136
121
257
43
16
59
R1
R2
51
30
81
33
31
64
41,2
56.7
46.9
60.6
17 54.8
37 57.8
1
2.6
4 10.0
4 13.3
8.3
9
19
16
35
18
17
35
97
120
217
92
157
249
40
10
50
44
14
58
R1
R2
M1
M2
M3
38
40
30
108
M1
M2
M3
35
41
31
107
Total
R2
No. of
pods
harvested
33
35
68
Total
Rl
Pod set
No.
%
R1
R2
Total
M2
Crossability between mung bean and rice bean
Total
21
17
38
20
0
2
1
3
Pods shed within
3 weeks.
4.9
3.2
2.8
X2 _ test for pod set and developed seeds
Comparison
M X R
vs. R X M
Within M X R
between M
between R
Within R X M
between R
between M
Pod set
38.43**
6.00ns
1. 26ns
0.86ns
22.08**
2.75 ns
4.93*
Developed seeds
172.34**
26.00**
27.03**
X2 value significant at 5% (*) or 1% (**); ns = not significant.
47
On mung bean, no difference in pod set between parental lines was
found.
Pods developed normally for 3 weeks, then dried prematurely
during the fourth week.
The dried pods contained two kinds of seeds,
empty and partially filled.
The empty seeds did not germinate, but an
average of two-thirds of the partially filled seeds germinated.
Seed
fertility differed between different combinations of parental lines.
The number of partially filled seeds obtained per pollination was 0.9
on Ml, 0.6 on M2 and 0.9 on M3.
This number was 1.1 in pollinations
by Rl, which is much higher than 0.4 by R2.
When rice bean was used as the female parent, pod set differences
between the two rice bean lines were not significant, but the differences in pod set effected by the three mung bean male parents were
significant.
Pods on rice bean usually shed within 3 weeks after
pollination.
Embryos older than 10 days were highly disorganized
sponge-like bodies.
The reciprocal difference in crossability implies that cytoplasmic
factors are responsible for the unidirectional success in this species
cross.
The isolating mechanism of hybrid inviability results in
complete embryo abortion on rice bean and in formation of partially
filled or empty seeds on mung bean.
Embryo culture
Since no seeds matured in the rice bean X mung bean crosses,
young embryos from these crosses were cultured.
Out of 20 11- to 23-
day old embryos cultured, none developed into a normal seedling
(Table 13).
Only one embryo formed a shoot without roots.
Deformed
48
Table 13.
Results of culturing hybrid embryos from
the cross between rice bean and mung bean
Female
Male
Age of
embryo
(days)
Rl
Ml
23
dried
1
deformed leaves
only
Rl
M2
12
green
3
2 callus
1 no growth
19
wilting
4
1 deformed leaves
and upright root
with tumored tip,
and callus
1 callus
2 no growth
23
wilting
2
2 callus
11
green
3
17
green
5
2 callus
1 deformed leaves
with callus
1 seed ling wi th
no root
4 no growth
21
wilting
2
Parents
Rl
R2
M3
M2
State
of
pod
No.
of
embryos
Results
2 callus
49
primary leaves developed in two embryos.
The extremely rare and
abnormal development of embryos in culture is additional evidence of
reciprocal differences in the strength of barriers to interspecific
gene exchange.
Plant growth and morphology of the hybrids
A characteristic feature of hybrids from mung bean X rice bean
was seedling weakness.
Hybrid seedlings were very weak and grew very
slowly at first (Fig. 19).
Abnormal seedlings with three or four
primary leaves often appeared.
The hybrid plants, however, passed
this weak seedling stage and grew very vigorously with thicker stems
and larger leaves than the parental species.
The hybrids were intermediate between the two parental species
in germination habit, petiole length of the primary leaves, color of
epicotyl, color of leaves, days to flower, and flower size.
The rice
bean characters of indeterminate growth, simple raceme, and glabrous
young pods were dominant in the Fl.
The edge of the standard was
purple and the upper part of the keel was greyish as in mung bean.
Trifoliate leaflets are entire in mung bean and often lobed in rice
bean, but the hybrids had lobed leaflets only occasionally (Table 14).
The hybrids were multi-branched sub-erect bushes during the
winter and vines in the summer.
Flowering occurred continuously in
the same racemes for an extraordinarily long period (Fig. 20).
hybrids grew as perennial plants, repeatedly produced juvenile
branches, and showed no senescence.
The
50
Figs. 19-20. The hybrids of mung bean X rice bean:
(Ml X Rl); 20) mature plants (M3 X Rl).
19) seedlings
Figs. 21-23. Meiosis in mung bean X rice bean hybrids: 21) 711 +
81; 22) llV + 311 + 121; 23) one polyspore of four larger and
three smaller microspores. Figs. 21-22 Xl,200 and Fig. 23
X480.
.
••
•
•
•
• •
-~
21
v
•
23
•
•
•
v,
••
•
52
Table 14.
Characters
Comparison of mung bean, rice bean,
and the hybrids between them
Mung bean
(females)
Hybrids
Rice bean
(males)
Germination
epigeal
intermediate
hypogeal
Primary leaves
sessile
short -petio1ed
long-petioled
(23 - 35 nun)
(2 - 6 nun)
Color of upper
epicoty1
green
light purple
purple
Color of leaves
dark green
intermediate
light green
Margin of
leaflets
entire
occasionally
lobed
often lobed
Growth habit
determinate
indeterminate
indeterminate
Flowering date
(planted on
5/7)
M1: 6/25
M2: 6/12
M3: 6/8
6/23 - 7/14
Rl: 9/21
R2: 6/21
Raceme
often compound
simple
simple
Edge of the
standard
purple
purple
bright yellow
Width of the
standard (nun)
17 - 20
24 - 26
23 - 25
Top of the keel
greyish
greyish
brigh t yellow
Young pods
hairy
glabrous
glabrous
Per cent
stainable
pollen
Ml: 96.5
M2: 95.7
M3: 91.5
0.8 (Ml X Rl)
to
5.8 (M3 X Rl)
Rl: 95.1
R2: 94.4
53
Fertility of the hybrids
A total of about 150 hybrid plants, grown in both winter and
summer, flowered profusely and set a few pods.
dropped in a week and never reached maturity.
The young pods, however,
Pistils of opened
flowers had very few or no pollen grains on them.
Percentages of
stainable pollen were 0.8 in the Fl of Ml X Rl, 0.9 in the Fl of Ml X
R2, 1.6 in the Fl of M2 X R2, 5.8 in the Fl of M3 X Rl and 1.7 in the
Fl of M3 X R2 compared to over 90% for the parental species.
The
pollen grain sizes were highly variable.
Numerous controlled self-pollinations and backcrosses to the parental species in every possible direction failed to produce any seeds.
Complete sterility of the hybrids operates as an absolute reproductive barrier between these two species at the diploid level.
Meiosis in the hybrids
Meiosis in the Fl plants was highly irregular.
In metaphase I,
the mean chromosome pairing was 0.09IV + 4.6911 + 12.261.
Maximum
pairing was 711 + 81 or lIV + 511 + 81 and modal pairing was 511 + 121
(Figs. 21 and 22), which are much lower than the maximum 1111 and
modal 811 + 61 reported by Dana (1966).
The frequency of the dif-
ferent chromosome configurations observed in the Fl hybrids is presented in Table 15.
The quadrivalents observed during meiosis, as Dana (1966) pointed
out, indicate that at least one reciprocal translocation has occurred
during chromosomal differentiation of these two species.
The lack of congregation of the bivalents and random distribution
of the univalents resulted in unequal separation of chromosomes at
54
Table 15.
Hybrids
Meiotic analysis of hybrids of mung bean X rice bean
No. of cells
examined
10
9
M1 X R1
Chromosome configurations
IV
II
I
Average
6
5
4
3
4.83
10
12
14
16
12.33
Average
6
3
5
2
4
3
4.52
7
Total
4
30
2
3
1
7
1
7
M1 x R2
Total
M2 X R1
Total
M2 X R2
Total
M3 X R1
Total
M3 X R2
2
3
3
21
1
2
3
1
1
8
2
1
3
1
9
4
20
1
8
4
1
9
1
1
25
1
2
1
1
1
0.14
7
Average
0.20
4.05
0.09
4.69
12.26
1
0.10
1
7
1
0.08
1
1
1
7
Total
Grand
total
5
20
124
6
3
5
4
3
Average
5.60
5
4
6
2
4
3
Grand
average
1l.75
10
12
12
14
14
16
13.60
8
8
10
12
12
14
16
10.48
8
10
10
14
14
16
13.10
1
Average
6
5
4
3
5.13
6
3
5
2
4
3
4.00
5
8
10
12
12
14
14
16
12.38
8
10
12
14
16
55
anaphase I.
The second division was also abnormal.
Chromosomes in
metaphase II failed to orient properly, which resulted in random
distribution of the second anaphasic chromosomes.
Polyspory was another peculiarity in microsporogenesis in these
hybrids, as was also reported by Sawa (1975).
The number of micro-
spores formed from a microsporocyte was highly variable.
Monads,
diads, and triads alone or together with up to six extra micronuclei
were observed at varying frequencies (Fig. 23).
Considering the extremely irregular chromosome associations in
both meiotic divisions and the polyspory observed, the production of
a gamete with a complete chromosome set of one genome would hardly be
expected.
Therefore, the complete sterility of the hybrids is ap-
parently due to the lack of chromosomal homology and resultant
meiotic irregularities, as Dana (1966) already pointed out.
AmphidiplOid
Thirty vigorous cuttings were taken from the completely sterile
Fl plants and treated with colchicine.
The colchicine-treated
cuttings rooted well and grew vigorously when transplanted to plastic
pots.
Of these 30 plants, 17 (56.6%) produced fertile branches which
yielded ripe pods with crinkled seeds.
When the crinkled seeds were
soaked in water, most appeared normal and germinated.
Some of the
seeds did not absorb water readily and germination was delayed.
The seedlings grew virorously with no seedling weakness as in
the original diploid hybrids (Fig. 24).
The general morphological
characters and plant growth were very similar to the diploid hybrids.
56
Figs. 24-25. Mung bean (M2) X rice bean (R2) amphidiploid (e2)
plants: 24) seedlings; 25) mature plants.
Figs. 26-29. Meiosis in the mung bean X rice bean amphidiploids:
26) diakinesis with 22 bivalents (arrow indicates 2 bivalents
superimposed in the picture); 27) metaphase with 22 bivalents;
28) metaphase II showing equal separation of chromosomes;
29) telophase showing equal distribution of chromosomes into
four microspores. Figs. 26 and 29 X480 and Figs. 27 and 28
Xl,200.
-flO- ..... ...,..
,
•
•
~
..... .. '..
..'" \.. •
~.
.
•
'28"
29
!
58
The plants grew very profusely as multi-branched semi-erect bushes
during the winter (Fig. 25) but as vines during the summer.
They began
to flower about the same time as the diploid hybrids and were perennial.
The gigas effect of polyploidy was observed in the floral
characters.
The width of the standard increased to 27 - 30 mm from
24 - 27 mm in the F1 hybrids.
The flower color was darker yellow and
the petals were thicker.
Pollen stainability increased to as much as 75.5 to 83.6% and
seed number per pod averaged 4.2.
increased later.
Pod set was very low at first, but
The seeds were crinkled like the ones produced by
the colchicine-treated plants (Cl).
Numerous crosses with the parental
species and the F1 hybrids failed to yield any pods.
Meiosis in the amphidiploids showed 22 biva1ents (Figs. 26-27)
and orderly disjunction in both anaphases (Figs. 28-29), similar to
that reported by Sawa (1975).
ation were grown.
Amphidiploid progenies up to C4 gener-
Plants with plump seeds or increased pod set were
not observed.
The complete sterility of the hybrids of mung bean X rice bean
can be effectively overcome by chromosome doubling, indicating that
sterility in F1 diploids is due largely to chromosomal factors.
The
crinkled seeds and low pod set may mean that some adverse genic interactions exist and thus these amphidip10ids are unlikely to become an
economic crop without further extensive breeding work.
However, their
exceptionally vigorous vegetative growth with strong perennialism and
substantial seed production may be useful for a soil-building or
forage crop.
59
5.
Crosses between black gram and mung bean
Crossability
This cross was successful only when mung bean was used as the
female parent.
Pod set was 29.4% on black gram and 58.6% on mung bean,
only slightly lower than for intraspecific pollinations.
Therefore, it
seems that there are no barriers to pod set between these two species.
The results of the crosses are presented in Table 16.
On black gram, pod set varied according to the combinations of
parental lines used.
The B1 X M2 cross produced the highest pod set
(46.0%) among six cross combinations.
but dried prematurely.
Pods seemed to develop normally
Dried pods contained two kinds of seeds:
and slightly filled seeds.
Neither kind of seeds germinated.
empty
Some
embryos appeared to be fairly normal, but the radicle and growing
point were poorly developed and the cotyledons were distorted and
fluffy.
On mung bean, differences in pod and seed set were highly significant between mung bean lines.
M2 had the lowest pod set (43.3%)
as well as the lowest seed number per pod (6.1) among the three lines.
No effect of black gram pollen on pod set was noticed.
veloped normally up to maturity.
The pods de-
Ripe pods contained partially
filled seeds, which appeared normal when soaked in water and germinated well.
There seem to be no barriers to fertilization between these two
species, at least in Some cross combinations of lines of the two
species.
However, there are barriers to seed development which result
60
Table 16.
Parents
Female Male
B1
No.
Seeds obtained
Empty
%
Filled
22.2
45.9
24.2
31.1
13
31
12
56
19
14
44
M1
M2
M3
30
35
30
95
11
6
9
26
36.7
17.1
30.0
27.4
37
7
7
51
8
8
12
28
B1
B2
32
30
62
23
21
44
71.9
70.0
71. 0
191
171
362
B1
B2
32
28
60
13
13
26
40.6
46.4
43.3
83
75
158
B1
B2
33
32
65
22
17
39
66.7
53.1
60.0
207
173
380
Total
M3
Pods harvested
8
17
8
33
Total
M2
No. of
flowers
crossed
black gram and mung bean
36
37
33
106
Total
M1
bet~een
M1
M2
M3
Total
B2
Crossability
Total
11
XL test for pod set and developed seeds
Comparison
B X M vs. M X B
Within B X M
between B
bet\'Jeen M
Within M X B
between M
between B
Pods harvested
Filled seeds obtained
9.56**
18.92**
0.24ns
0.43 ns
14.99*'/'
6.67**
0.08 ns
383.75**
156.47**
0.007 ns
19.65**
295.07**
143.85**
0.94ns
**significant at 1% level; ns
= not
significant.
61
in empty and nonviable seeds on black gram and slightly defective but
viable seeds on mung bean.
Embryo culture
Fourteen hybrid embryos on black gram were cultured.
these embryos developed into normal seedlings.
None of
As shown in Table 17,
three embryos formed callus only and others did not show any sign of
growth.
Thus, incompatibility between the degenerating embryos and
the developing endosperm does not seem to be the cause of failure of
this cross.
The reciprocal difference in crossability between black gram and
mung bean suggests that a disharmonious interaction between genic and
cytoplasmic factors may be responsible for the embryo degeneration
when the cross is made on black gram.
Plant growth and morphology of the hybrids
There was a difference in the vitality of hybrids from different
lines of mung bean.
The hybrids displayed hybrid vigor in their early
seedling growth (Fig. 30).
Vigorous plant growth extended throughout
the growing season in the hybrids of Ml and M2.
However, the hybrids
from crosses with M3 as females (M3 X Bl and M3 X B2) suddenly lost
vigor upon forming flower buds and barely grew from then on.
The hybrids resembled mung bean in having a purple-tinged edge
of the standard and grey color in the upper part of the keel; resembled black gram in having a simple raceme, long-hairy and upright
pods, ovoid seeds, and a concave hilum cushion; and were intermediate
in foliage color (Table 18).
62
Table 17.
Parents
Results of culturing hybrid embryos from the
cross between black gram and mung bean
Female
Male
Age of
embryo
(days)
B1
M2
11
State
of
pod
No. of
embryos
Results
green
green
1
1
no growth
no growth
14
green
green
3
1
3 callus
no growth
16
B1
M3
11
B2
M2
16
35
green
dried
4
1
no growth
no growth
B2
M3
13
green
3
no growth
63
Figs. 30-31. The hybrids of mung bean (M2) X black gram (B2):
30) seedlings; 31) mature plants.
Figs. 32-35. Meiosis in mung bean X black gram hybrids: 32) metaphase I showing 11 bivalents; 33) diakinesis - lIV + 811 + 21
(univalents are indicated by arrows); 34) diakinesis - lIV +
911; 35) one polyspore of five microspores. Figs. 32-34 Xl,200
and Fig. 35 X480.
Fig. 36. A Bel plant from mung bean X (mung bean X black gram) (M2 X
Fl of M2 X B2), showing increased pod set.
31
•
,.,
\
,-
I'
.t
r·
32
{
.l.
II'
33
.......::.
34
.35
65
Table 18.
Morphological comparison of mung bean,
black gram, and the hybrids between them
Nung bean
(females)
Hybrids
Color of leaves
dark green
intermediate
light green
Raceme
often compound
simple
simple
Edge of the standard
purplish
(M2, M3)
purplish
dark yellow
Top of the keel
greyish
greyish
dark yellow
Hairs on pod
short
long
long
Pods at maturity
radiate or
drooping
upright
upright
Shape of seed
globose
ovoid
ovoid
Color of seed
green
brown and
black spotted
dark grey and
white spotted
Hilum cushion
not concave
concave
concave
Per cent
stained
pollen
96.5 (Ml)
95.7 (M2)
91.5 (M3)
24.0 (M3 X B2)
to
26.6 (Ml X B2)
95.9 (Bl)
95.1 (B2)
Characters
Black gram
(males)
66
Plant growth was indeterminate like black gram; multi-branched
with slight tendency to vine in the winter and viny during the summer.
The F1 hybrids of M3 X black gram also had indeterminate growth, but
did not become viny in the summer because of the severely weakened
plant growth, and died after a short period of fruiting.
Fertility of the hybrids
The hybrids were partially fertile.
The hybrid plants flowered
profusely but pod set was very low (Fig. 31).
A few ripe pods har-
vested from open-pollinated flowers contained usually one or rarely
two seeds.
veloped.
The seeds were of various kinds, from empty to fully deSome seeds had burst seed coats.
from 24.0% to 26.6%.
Pollen stainability ranged
The stained pollen grains were comparatively
uniform, but the unstained grains were much smaller and highly variable in size.
Viable seeds were produced when the hybrids were used as both
male and female parents in backcrosses to the parental species.
Meiosis in the hybrids
Many kinds of irregularities were observed during microsporogenesis in the hybrids.
Chromosome configurations at diakinesis and
at metaphase 1 are presented in Table 19.
Complete bivalent formation
occurred in 28.6% of 147 cells examined (Fig. 32).
The other cells
contained one quadrivalent and/or two or four univa1ents (Figs. 32 and
34).
The mean chromosome configuration was O.371V + 9.5911 + 1.341.
Often the bivalents failed to orient properly on the equatorial
plate.
Heteromorphic pairing was noticed in diakinesis.
Chromosome
67
Table 19.
Hybrids
M1 X Bl
Ml X B2
Meiotic analysis of hybrids of mung bean X black gram
No. of cells
examined
5
7
2
5
1
5
Total
25
1
4
2
10
1
6
24
Total
Chromosome configurations
IV
1
M2 X B1
Total
M2 X B2
Total
M3 X B1
Total
M3 X B2
Total
Grand
Total
147
I
11
1
1
Average
0.32
1
8
10
7
9
9.60
9
2
2
4
4
1,52
11
1
1
Average
11
13
1
1
26
4
7
6
7
1
2
27
5
6
7
6
1
2
27
4
5
2
3
1
3
18
II
9
0.17
1
8
10
7
9
9.58
9
2
2
4
4
2.16
11
1
Average
0.46
1
8
10
10.00
9
2
2
0.16
11
1
1
Average
0.41
1
8
10
7
9
9.48
9
2
2
4
4
1.40
11
1
0.48
1
8
10
7
9
9.33
9
1
11
8
1
Average
0.39
10
7
9
9.50
2
2
4
4
1,44
0.37
9.59
1.34
1
Average
Grand
Average
2
2
4
4
1.41
68
bridges occurred in 3 cells and 1 to 5 laggards were observed in 5 cells
out of 29 cells examined at anaphase I.
anaphase I was studied in 21 cells.
Chromosome distribution at
Chromosomes had separated equally
11-11 in 11 cells, 10-12 in 9 cells, and 13-9 in one cell.
Polyspory
was observed in many tetrads (Fig. 35).
The results show that these two species are closely related with
chromosome differentiation having occurred through one major reciprocal translocation.
This confirms the reports of Dana (1966a), and
De and Krishnan (1966b).
The low pod set compared with the fairly
high frequency of complete bivalent formation indicates that the cause
of the sterility may be partly due to either chromosomal heterozygosity or genic interaction.
From pachytene chromosome analysis, De
and Krishnan (1966) reported that a terminal deletion and an interstitial duplication are also involved.
The F? and backcross generations
Germination of well developed seeds from Fl plants was highly
irregular.
Seeds germinated sporadically over an extended period.
Some seeds remained hard and germinated late and others failed to
germinate at all.
(Table 20).
Out of 74 seeds, 47 (60%) germinated in 10 days
Germination in 3 days was quite uniform in mung bean,
black gram and the Fl.
Seedling lethality and weakness were other characteristics found
in the F2 generation.
Fifteen seedlings died during early or late
seedling stages, among which three were complete albinos, four were
sectorial albinos, and the rest died after forming clusters of curly
leaves or when flower buds formed.
The hybrid vigor of the Fl was
69
Table 20.
The F2 generation of mung bean X black gram crosses
Crosses
No. of
seeds
sown
Germinated
seeds
No. of
plants
flowered
M1 X B1
13
9
7
9.8-51. 5
1
M1 X B2
13
7
5
12.1-54.3
2
M2 X B1
13
8
6
16.1-71.6
5
M2 X B2
13
8
5
11. 9-68. 6
3
M3 X B1
12
8
5
8.3-69.5
4
M3 X B2
10
7
4
17.0-57.6
3
Total
74
47
32
8.3-71. 6
18
%
stained
pollen
No. of
plants
set pods
70
completely lost in the F2 generation.
No individual F2 plant grew as
vigorously as the F1 plants.
Wide variability in fertility was observed in the F2 generation.
Pollen stainability ranged from 9.26 to 71.63% (Table 20).
Eighteen
out of 32 plants which flowered produced pods with well-developed
seeds.
All combinations of backcrosses to the parental species produced
viable seeds (Table 21).
No differences in pod set between the back-
crosses to mung bean and black gram were found.
However, differences
due to the direction of the crosses to each parental species were
highly significant.
In backcrosses to black gram, pod set and seed
production were much higher when the F1 plants were used as the females.
Pod set was significantly higher on F1 plants in backcrosses
to mung bean, but there was no difference in seed number, because of
the higher seed number in the mung bean pod.
Hybrid lethality was much lower in the backcross progenies (BC1)
than in the F2.
Out of 38 seeds, 27 (70%) seedlings were obtained, of
which 3 died during seedling stages, 3 did not flower, and 21 flowered.
All of the BCl plants showed a mixture of mung bean and black gram
characters.
Plant growth was highly diverse, from dwarf to normal
growth, and from bush to vine.
progenies.
as 75.1%.
Pollen stainability ranged from as low as 4.0% to as high
Three plants with less than 20% stainable pollen did not
produce any mature pods.
(Fig. 36).
Fertility varied in the backcross
Pot set was very high in some plants
71
Table 21.
Crosses
Female Male
M1B1
(F1)
M1
B1
M1B2
(F1)
M1
B2
M2B1
(F1)
M2
B1
Backcrosses of hybrids of mung bean X
black gram to their parents
No. of
flowers
crossed
Pods harvested
No.
%
No. of
seeds
obtained
Progenies grown
% stained
No.
pollen
M1
B1
14
11
2
2
14.3
18.2
2
4
1
2
27.5
no flower
M1B1
(F1)
10
10
1
1
10.0
10.0
2
1
2
1
38.6, 67.4
41.9
Ml
B2
11
13
0
1
7.7
1
1
25.1
M1B2
(F1)
10
15
0
1
6.7
1
1
no flower
M2
B1
16
17
5
7
31. 3
41.2
5
5
3
3
30.3-63.6
33.0-75.1
M2B1
(F1)
12
10
0
0
M2B2
(F1)
M2
B2
M2
B2
13
13
1
2
7.7
15.4
1
1
0
0
M2B2
(F1)
12
10
1
0
8.3
4
3
6.36-66.0
M3B1
(F1)
M3
B1
16
17
3
6
18.8
35.3
1
3
1
2
50.3
30.0, 54.3
M3B1
(F1)
15
10
1
0
6.7
5
3
15.9-64.8
M3
B2
8
5
0
2
40.0
2
1
M3B2
(F1)
7
2
0
0
M3
B1
H3B2
(F1)
M3
B2
4.0
X2_ test for pod and seed set
Comparison
BCM vs. BCB
F1 X B vs. B X F1
F1 X M vs. M X F1
Pods
Seeds
1. nns
17.44**
4.89*
0.005ns
12.53**
0.93ns
X2 value significant at 5% (*) or 1% (**); ns
= not significant.
72
Germplasm exchange between mung bean and black gram is highly
possible through backcrosses to either parent.
6.
Crosses between adzuki bean and rice bean
Crossability
When adzuki bean was used as the female, there was no significant
difference in pod set between the two lines of adzuki bean (Table 22).
The difference between the two rice bean lines used as pollen parents
was also not significant.
The overall pod set in these two crosses
was 55.6% which is significantly higher than the 23.0% pod set for
intraspecific crosses on adzuki bean.
Pods developed normally and
reached their maximum size in 3 weeks, when they suddenly discolored,
lost turgidity, and collapsed.
Pods older than one week contained
only highly distorted, sponge-like embryos.
When rice bean was used as the female, significant differences
were found between parental lines of both species.
R2 set more pods
(31.3%) than Rl (10.8%) and A2 was a better pollinator (27.3%) than
Al (12.7%).
The highest pod set (36.6%) was from the cross R2 X A2.
Pods developed slowly and were shed 2 to 3 weeks after pollination.
Embryos from pods 11 days old or more appeared normal and healthy.
Shrivelled seeds obtained from dried pods appeared normal when soaked
in water but failed to germinate.
Apparently, there is no barrier to fertilization in adzuki bean X
rice bean crosses.
The higher pod set on adzuki bean in the crosses
with rice bean is most likely due to scant pollen production on adzuki
bean during the winter.
However, in rice bean X adzuki bean crosses,
73
Table 22.
Crossability between adzuki bean and rice bean
No. of
flowers
crossed
Parents
Female Male
Al
33
16
48.5
R2
32
20
62.5
65
36
55.4
Rl
32
20
62.5
R2
38
19
50.0
70
39
55.7
Al
29
1
3.4
A2
36
6
16.7
65
7
10.8
Al
26
6
23.1
A2
41
15
36.6
67
21
31.3
Total
Rl
Total
R2
%
pod set
Rl
Total
A2
No. of
pods set
Total
2
X - test for pod set
Comparison
A X R vs. R X A
X2 value
Within A X R
between A
between R
15.37**
3.17 ns
O.OOlns
O.OOlns
Within R X A
between R
between A
28.56**
10.05**
5.29*
X2 value significant at 5% (*) or 1% (**); ns
not significant.
74
the differential pod set on Rl and R2 indicates that barriers to
fertilization vary according to rice bean lines used in the crosses.
Extremely early degeneration of embryos when adzuki bean was used
as the female was another directional difference.
The presence of
normal pod development suggests that growth substances promoting fruit
development were present after degeneration of the embryos.
Embryo abortion and failure of partially filled seeds to germinate are effective barriers to gene exchange between adzuki bean and
rice bean.
Embryo culture
Of 17 embryos 8 to 11 days old cultured from adzuki bean X rice
bean crosses, none developed into normal viable seedlings (Table 23).
One embryo developed abnormal primary leaves with callus, and three
others callused only.
The remainder of the embryos failed to ex-
hibit any sign of growth.
Forty seven embryos 11 to 24 days old from rice bean X adzuki
bean crosses were cultured.
As shown in Table 23, viable seedlings
were obtained only from the cross R2 X A2.
Twelve pale green and 12
healthy seedlings developed out of 33 embryos cultured.
All the pale
green seedlings and five of the healthy seedlings died during seedling
stages, while the other seven healthy seedlings reached maturity.
The exact cause of the seedling etiolation is uncertain, but it
is evident that normal, viable hybrids can be secured in certain rice
bean X adzuki bean crosses by embryo culture.
The reciprocal difference in hybrid embryo development in these
crosses implies that cytoplasmic differentiation is a principal factor
75
Table 23.
Parents
Female Male
Results of culturing hybrid embryos from crosses
between adzuki bean and rice bean
Age of
embryo
(days)
State
of
pod
No.
of
embryos
Results
Al
Rl
18
green
1
no growth
Al
R2
13
green
1
no growth
A2
Rl
8
10
green
green
3
3
no growth
1 callus
2 no growth
16
green
3
no growth
10
14
green
green
2
2
15
green
2
no growth
1 callus
1 no growth
1 deformed leaves with
callus
1 no growth
15
wilting
3
no growth
11
wilting
wilting
2
16
4
no growth
4 etiolated seedlings
14
dropped
3
22
green
1
12
green
5
18
green
5
20
21
21
green
green
wilting
4
3
10
21
dropped
green
green
dried
1
2
1
2
A2
R2
Rl
Al
Rl
A2
R2
R2
Al
A2
22
24
24
1 etiolated seedling
2 no growth
etiolated seedling
2 mature hybrids
3 seedlings died
1 mature hybrid
4 seedlings died
4 etiolated seedlings
3 etiolated seedlings
8 etiolated seedlings
2 no growth
1 mature hybrid
2 mature hybrids
1 mature hybrid
no growth
76
involved in the evolution of isolating mechanisms between these two
species.
Morphology of the hybrid
The shape of the primary leaves of the hybrid was ovate (Fig. 37)
as compared to linear lanceolate in rice bean and broadly cordate in
adzuki bean (Table 24).
The hybrids resembled the female species, rice
bean (R2), in many ways.
The rice bean characters of elongated raceme,
blackish-grey color of the ripe pod, dark yellow flower color, greenishyellow seed color, and concave hilum cushion appeared in the hybrids.
The constriction of the pod between the seeds and the shape of the
seeds were intermediate between the two parents.
The green foliage
color and erect type of growth of the adzuki bean were dominant
(Fig. 38).
Trifoliate leaflets were lanceolate in the hybrids, but
rhombic in both parents.
The hybrids produced much heavier seeds than
either adzuki bean or rice bean.
The length of pod and number of seeds
per pod were reduced in the hybrids.
The reduction of pod length and
seed number is perhaps the result of reduced fertility in the interspecific hybrid.
Fertility of the hybrid
The hybrids produced 76.0% stainable pollen as compared to 94.4%
in R2 and 91.0% in A2.
The number of seeds per pod was 3.2 which is
52.5% of the midparent value (6.0).
Viable seeds were produced in
backcrosses to both parental species in all combinations.
It seems that once embryo abortion is circumvented by embryo
culture no other reproductive isolation barrier exists to restrict gene
exchange between these two species.
77
Figs. 37-38. The hybrids of rice bean (RZ) X adzuki bean (AZ)
obtained by embryo culture: 37) seedlings; 38) mature plants.
Figs. 39-41. Regular meiosis in rice bean (RZ) X adzuki bean (AZ)
hybrid: 39) metaphase I - 1111; 40) metaphase II; 41) telophase. All figures Xl, ZOO.
Figs. 4Z-43. FZ plants of rice bean (RZ) X adzuki bean (AZ):
4Z) seedlings showing segregation for the shape of primary
leaves; 43 mature plants with good pod set.
..
•
.JI
. . . . . ,.1
',.
•
:;:'
/,~.,.;~-:~/fj?~,..'.:~~,
i~.,~ ~.,·.~".:,.· ·,·~.if; .'
·....,
,. .'. ,:..:,.'. . ,.
7' " -
."l1·f.....
39
_i
.''!'
.'
;'.~
I
41
."
. ("
.
.II
.t.-
79
Table 24.
Comparison of rice bean (R2), adzuki bean (A),
and the hybrid between them
Adzuki bean
(male)
Characters
Rice bean
(female)
Shape of primary
leaves
linear
lanceolate
ovate
broadly
cordate
Color of epicotyl
purple
light purple
light green
Color of leaves
light green
green
green
Shape of leaflets
rhombic
lanceolate
rhombic
Constriction of pod
smooth
intermediate
conspicuous
Plant growth
sub-erect,
multi-branched
erect,
simple stem
erect,
simple stem
Raceme
elongated
elongated
compact
Color of ripe pod
blackish grey
blackish grey
straw
Seed color
greenish
yellow
greenish
yellow
maroon
Hilum cushion
concave
concave
not concave
Seed size (mm)
length X width
7.1 X 4.1
6.9 X 4.5
5.5 X 4.2
Seed weight (mg)
7.5
9.0
6.6
Pod length (cm)
8.4
6.1
7.5
No. of seeds per pod
6.3
3.2
5.7
94.4
76.0
91.0
Pollen stainability
(%)
Flower color
bright yellow
Hybrid
bright yellow
light yellow
80
Meiosis in the hybrid
The hybrids regularly formed 11 biva1ents at first metaphase in 26
cells examined (Fig. 39).
As in the parental species, precocious sep-
aration of a few biva1ents was observed.
Otherwise the separation and
movement of the chromosomes in both anaphases was normal (Figs. 40 and
41).
The F2 and backcross generations
The seeds from Fl plants germinated normally.
In 23 F2 plants,
continuous variation was observed in the shape of the primary leaves
(Fig. 42) and epicoty1 color, indicating multiple genes are involved in
the inheritance of these characters.
for two dwarfs.
Plant growth was normal except
The dwarf plants grew slowly and took exceptionally
long to produce a few flowers.
Five plants had 1anceo1ate leaflets as
in the F1 and the remaining 16 plants had the rhombic leaflets of both
parental species.
The growth habit was an erect bush with a simple
main stem in 10 plants and sub-erect with multiple stems in 11 plants.
The red seed color of the adzuki bean was recovered in four plants.
Segregation for fertility was observed.
Stainable pollen ranged from
48.5 to 93.3% in five plants examined.
Plants with a high percentage
of stainable pollen also had good pod set (Fig. 43).
In backcrosses to the parental species, viable seeds were produced in all of the four possible combinations.
cient
f1owerin~
Because of insuffi-
of A2 at the right time, Al and the inter-line hybrid
(A2 X Al) were sometimes substituted for A2 in these backcrosses.
As shown in Table 25, reciprocal differences in the backcrosses to
adzuki bean are not significant, but reciprocal differences are highly
81
significant in the backcrosses to rice bean.
The extremely low pod set
on rice bean (8.3%) was probably caused by red spider mite damage during
fruit development.
Rice bean was severely damaged by the mites long
before adzuki bean and the F l hybrids showed visual injury.
A lower
rate of viable pollen in Fl plants probably caused lower pod set on
rice bean also.
The differences between the backcrosses to the
paternal and maternal species indicate that cytoplasmic factors may be
involved in seed failure in this species cross.
Progenies from all of the possible backcrosses were produced.
However, two plants from the A2 X Fl cross and one plant from the
(A2 X AI) X FI cross died before flowering.
The remaining plants
reached maturity and produced normal seeds.
Seed production of the
backcross progenies was directly proportional to the percentages of
their pollen stainability (Table 25).
The adzuki bean seed color,
which was recessive in the FI, was recovered in three out of five backcrosses to adzuki bean.
The plant characters of the backcrosses re-
sembled the parental species used in the cross, but plants with all
the characters of the backcross parental species did not appear.
This species hybridization indicates that embryo abortion due to
disharmonious interaction between cytoplasmic factors of one species
and genic factors of the other can be overcome only by culturing
developing embryos from the cross with rice bean as the female parent.
Success also seems to depend upon the particular combination of genotypes of the two species.
The high fertility of the FI, F2 and back-
cross generations indicates that no barrier other than embryo abortion
blocks the exchange of genes between these two species.
Therefore, it
82
Table 25.
Backcrosses of the rice bean (R2) X adzuki bean (A)
hybrid to the parental species
No. of
progenies
flowered
%
stained
pollen
Male
A2
5
2
40.0
1
1
50.1
Al
4
1
25.0
4
2
75.1
80.3
A2A1
10
1
10.0
1
0
19
4
21.1
6
3
6
3
50.0
5
0
A2A1
15
3
20.0
3
2
88.7
70.1
Total
21
6
28.6
8
2
79.4
R2
18
15
83.3
87
11
75.1
to
93.3
R2A2
F1
24
2
8.3
3
1
88.3
Female
R2A2
F1
Total
A2
R2A2
F1
R2
R2A2
F1
Pods
harvested
No.
%
No. of
seeds
obtained
No. of
flowers
crossed
Crosses
68.7
X2_ test for pod and seed set
Comparison
Pods
Seeds
Fl X A vs. A X Fl
3.66ns
1. 14ns
128.95**
0.61ns
Fl X R vs. R X F1
61. 37**
447.10**
BCA vs. BCR
**high1y significant; ns
not significant.
83
appears that these two species are very closely related and that an
isolation mechanism has evolved primarily through cytoplasmic differentiation resulting in embryo abortion.
Accordingly, once embryo
abortion is overcome, it would be possible to exchange germplasm
between these two species and thus improve either adzuki bean or rice
bean.
GENERAL DISCUSSION AND CONCLUSIONS
A variety of isolating mechanisms were found to operate between
Vigna radiata,
~.
umbellata,
~.
angularis and
~.
mungo.
The results
of the hybridizations among these species are summarized in Table 26.
Normal pod set means pod set in at least one cross was equal to that
found in intraspecific pollinations.
After pods had set, in most cases
the embryo aborted before reaching maturity.
Only in the pollinations
of mung bean by black gram and rice bean was there not complete embryo
abortion.
Immature embryos were cultured on artificial medium in all
pollinations where the embryos did not mature naturally.
In some cases,
successful embryo culture was followed by loss of all seedlings in the
seedling stage.
A further difference was noted in that some seedlings
which survived remained weak, while others grew well.
Finally, some
hybrids were completely sterile, while others were only partially
sterile.
These differences and the meiotic configurations found in the
hybrids are shown in Table 26.
Two or three lines of each species were used in the crosses.
The
variability in crossability shown by different lines of the same species
suggests that the use of more than one genotype as well as intraspecific
hybrids is desirable when making interspecific crosses.
Reciprocal differences in crossability were found in all interspecific crosses.
The differences were observed in pod set, embryo
abortion, embryo culture, and seedling lethality.
The results of the six species crosses are summarized as follows:
The cross between adzuki bean and black gram was the least successful.
Although pod set was normal when adzuki bean was used as the
Table 26.
Crosses
Summary of hybridizations among the four Vigna species
Pod
set
Embryo
abortion
Embryo
culture
Seedling
lethality
Adzuki bean X Black gram
normal
complete
partial
complete
Black gram X Adzuki bean
partial
complete
partial
complete
Black gram X Rice bean
normal
complete
partial
partial
Rice bean X Black gram
partial
complete
none
Adzuki bean X Mung bean
normal
complete
partial
complete
Mung bean X Adzuki bean
partial
complete
partial
partial
Mung bean X Rice bean
normal
partial
Rice bean X Mung bean
partial
complete
none
Black gram X Mung bean
normal
complete
none
Mung bean X Black gram
normal
none
Adzuki bean X Rice bean
normal
complete
none
Rice bean X Adzuki bean
normal
complete
partial
Female
Male
Hybrid
weakness
Hybrid
sterility
Hybrid
meiosis
none
complete
2. 39II+17.221
partial
none
complete
O.09IV+4.69II
+12.261
none
partial
partial
O.37IV+9.59II
+1.341
partial
partial
partial
llll
complete
CXl
VI
86
female, embryo abortion was complete and no seedlings obtained from
embryo culture survived beyond the seedling stage.
These two species
appear to be the most distantly related of the four species.
The cross between black gram and rice bean was also very difficult.
Some seedlings obtained by embryo culture from crosses on black
gram reached adult stage, but died before flowering.
Therefore, these
species are also considered to be very distantly related.
The cross between adzuki bean and mung bean was somewhat more
successful.
Hybrids from mung bean produced by embryo culture flowered
profusely but were completely sterile.
Lack of chromosome pairing,
shown by an average of 2.39 11 + 17.22 1 , seems likely to be a principal
cause of sterility.
These two species also seem to be distnat1y re-
lated.
The cross between mung bean and rice bean was somewhat easier
than the last.
Embryo abortion is not complete so that some crinkled
but viable seeds are produced on mung bean.
However, the hybrids, with
average chromosome pairing of O.091V + 4.6911
completely sterile.
+ 12.261' are still
Colchicine-induced amphidip10ids showed regular
meiosis with 22 bivalents and greatly improved fertility, indicating
that at least in this combination much of the sterility of the diploid
hybrid is caused by chromosomal factors.
However, low pod set and de-
fective seeds in the amphidiploid indicate that some adverse genic
interactions are still found.
These two species must be more closely
related than any of the previous combinations.
Black gram and mung bean seem to be very closely related.
Hybrid
seed development, when mung bean was used as the female, was almost
87
normal and germination was good.
The Fl hybrids were partially fertile.
One quadrivalent in 37% of the cells examined indicates that one reciprocal translocation has occurred during chromosomal differentiation of
these two species.
Segregation for morphological characters and the
appearance of highly fertile individuals in the F2 and BCl demonstrate
the possibility of gene exchange between these two species.
Adzuki bean and rice bean also seem to be closely related.
Hy-
brids obtained by culture of embryos from rice bean were highly fertile
with completely normal bivalent formation.
Segregation for morpho-
logical characters and high fertility in the F2 and BCl indicate the
possibility of gene exchange between these two species also.
Since unidirectional success is a common phenomenon ;.n all crosses
between any two species, cytoplasmic differentiation is assumed to have
played the primary role in speciation.
Cytoplasm which prevents the development of a hybrid embryo can be
considered to be an evolved cytoplasm, while cytoplasm which permits
development of a hybrid embryo can be considered to be the original
type.
By this reasoning, mung bean would have more nearly the original
cytoplasm, since this cytoplasm had by far the least inhibitory effect
on hybrid embryos.
y.
radiata var. soblobata (syn. Phaseolus soblobatus), which is a
wild species and considered to be the progenitor of mung bean and
black gram by Zukovskij (1962), is a likely common ancestor of these
four species.
are
p08tul~ted
The evolutionary relationships of these four species
in Fig. 44.
88
Progenitor
v.
radiata
mung bean
v.
mungo
black gram
Fig. 44.
v.
umbellata
rice bean
V. angularis
adzuki bean
Proposed evolutionary relationships of
the four Vigna species.
89
Mung bean and rice bean might have been derived independently from
this wild species, but the rice bean probably evolved first.
Black gram
and adzuki bean seem to be later derivatives from mung bean and rice
bean, respectively.
Thus, these four species can be divided into two
subgroups, mung bean with black gram and rice bean with adzuki bean, in
which the two species of each group retain enough chromosomal homology
to allow germp1asm exchange within the group.
This postulation is supported by the grouping of these four species
into two taxa by Piper and Morse (1914) on the basis of morphological
similarities.
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